This bridging proposal addresses a major opportunity in biological research to provide multifunctional 3D superresolution imaging capability to thousands of biology laboratories in the US and around the globe. The objective of this project is to develop, to the point of commercial production, a ground-breaking multifunctional microscopy system with single-molecule sensitivity suitable for live cell studies. The system overcomes current fundamental limitations by providing wide-field three-dimensional (3D) super-resolution imaging with single molecule sensitivity. These capabilities are achieved using minimally invasive fluorescence techniques. Intellectual merit--A variety of methods for super-resolution optical microscopy are now making it possible to resolve objects that are smaller than the optical diffraction limit, which has historically restricted spatial resolution to about 200nm in the transverse dimension and about 500 nm in the axial dimension. Although these techniques have demonstrated the feasibility of optical imaging beyond the diffraction limit, they are far from mature and new developments are required to reach the new limits on a regular basis at the typical biology lab. Accordingly, this project focuses on fundamental developments to address the need for widespread single-molecule imaging instrumentation. The instrument is based on an integrated design of the illumination, the fluorescent molecules, the 3D optical response, the data collection strategy, and the postprocessing / reconstruction algorithms. The proposed design of a wide-field microscope presents a double-helix point spread function that features two dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. By encoding the z-position in the angular orientation of the two lobes, the 3D position of each emitter can be determined well beyond the optical diffraction limit. Moreover, the technique enables 3D imaging with greater depth of field than is available from other imaging methods. The system consistently attains 3D sectioning capability with isotropic resolutions below 20nm over an extended depth of field of several microns, representing one order of magnitude improvement over available optical microscopes at most research institutions. A well thought-out dissemination plan provides a path from prototype development through large scale production. The main development tasks in this project are: (a) Bringing to maturity a scalable fabrication process for efficient double-helix phase masks. (b) Advancing the implementation of reconstruction algorithms for real-time operation and ease of use by biologists. (c) Implementing a flexible modular structure that can be integrated with new or existing microscopes. (d) Testing the instrument in significant biological problems and independent biology labs. Broader Impact - The widespread availability of 3D superresolution imaging will impact multiple fields of science and engineering including 3D biophysical and biomedical imaging of labeled biomolecules inside and outside of cells. The techniques will find use in tracking orientation changes of molecules within a cellular structure, as well as in monitoring interactions between molecules. The project integrates education and outreach with research and development to create the human infrastructure required for developing future biological instrumentation technologies. It will provide training to a diverse group of young scientists and engineers. Through interaction with a local IGERT program in computational optical sensing and imaging, this project will provide opportunities for interdisciplinary student rotations. The project will also generate synergistic relationships with industry to transfer new discoveries into industrial applications. The results of the investigation will be broadly disseminated including an outreach traveling exhibit on microscopy with interactive demonstrations.
